In the early 1990's Bloom and colleagues successfully rescued vasopressin-deficient rats by injecting in vitro-transcribed vasopressin mRNA into the hypothalamus (Science 255: 996-998; 1992). However, the low levels of translation and the immunogenicity of the molecules hampered the development of mRNA as a therapeutic and efforts have since focused on alternative applications that could instead exploit these pitfalls, i.e. immunization with mRNAs coding for cancer antigens.
Others have investigated the use of mRNA to deliver a polypeptide of interest and shown that certain chemical modifications of mRNA molecules, particularly pseudouridine and 5-methyl-cytosine, have reduced immunostimulatory effect. Notwithstanding these reports which are limited to a selection of chemical modifications including pseudouridine and 5-methyl-cytosine, there remains a need in the art for therapeutic modalities to address the myriad of barriers surrounding the efficacious modulation of intracellular translation and processing of nucleic acids encoding polypeptides or fragments thereof.
To this end, the inventors have shown that certain modified mRNA sequences have the potential as therapeutics with benefits beyond just evading, avoiding or diminishing the immune response. Such studies are detailed in published co-pending applications International Application PCT/US2011/046861 filed Aug. 5, 2011 and PCT/US2011/054636 filed Oct. 3, 2011, International Application number PCT/US2011/054617 filed Oct. 3, 2011, the contents of which are incorporated herein by reference in their entirety.
The present invention builds upon the aforementioned disclosures and provides methods and compositions useful in improving the efficiency of in vitro transcription and translation systems supporting the discovery and development of chemically modified messenger RNA.
Specifically, the present invention utilizes the PACE (phage-assisted continuous directed evolution) method described by Liu for the production of novel enzymes and enzyme variants currently used in in vitro transcription and post-transcriptional systems (Esvelt et al. (Nature (2011) 472(7344):499-503 and U.S. Publication No. 20110177495). These new enzymes have properties which allow for the production of a broader array of modified messenger RNA molecules, especially those which incorporate unnatural chemical modifications which previously could not be incorporated into an mRNA during in vitro transcription.